As the second most expressed membrane receptor complex on the platelet surface, the glycoprotein (GP) Ib-IX-V complex is essential to platelet physiology. It is identified primarily as the receptor for a number of hemostatically important proteins including von Willebrand factor and thrombin. It has also been implicated in the genesis and clearance of platelets. Malfunction of this multi-subunit receptor complex can lead to severe bleeding diathesis and contribute to many cardiovascular diseases. However, how this complex carries out its multi-faceted functions is not clear, primarily due to the lack of understanding of its structure and organization. The GPIb-IX-V complex comprises of 4 different type I transmembrane proteins, with the GPIb-IX complex possessing the ligand-binding activity and signaling capability. We hypothesize that inter-subunit interactions are important to the functions and regulation of the GPIb-IX complex. In this project, we aim to elucidate its organizing principle and to explore functional roles of inter-subunit interactions. In Specific Aim 1, to characterize the postulated weak interaction between the ectodomains of GPIb1 and GPIX subunits, we will define the interfacial region and residues in both domains and determine their roles in the assembly and inside-out regulation of the receptor complex. Specific Aim 2 is to elucidate the structural basis for the inter-ectodomain interactions. Structure of the recombinant GPIb2 ectodomain will be determined by X-ray crystallography. Engineered GPIX variants with improved stability that retain the native binding region to GPIb2 and GPIb1 will be produced for structural determination and functional analysis. Specific Aim 3 is to elucidate the structural basis for the interaction among transmembrane helices in the GPIb-IX complex by heteronuclear triple resonance NMR spectroscopy. Overall, careful biochemical and structural dissection of individual interactions in isolated protein domains, in transfected mammalian cells and in human platelets will help to elucidate the likely structural changes in the GPIb-IX complex in response to intracellular regulatory signals and ligand binding, and to provide insights on the molecular basis for GPIb-IX-related diseases. This project will also afford us the opportunity to develop tools and reagents to specifically perturb the inter-subunit interaction of interest, which may lead to novel therapeutic strategies.